Int. J. Morphol., 34(4):1419-1423, 2016. Characterization of the Campylobacter fetus subsp. venerealis Adhesion to Bovine Sperm Cells Caracterización de la Adhesión de Campylobacter fetus subsp. venerealis a Espermatozoides Bovinos María L. Chiapparrone*; Pedro Soto* & María Catena* CHIAPPARRONE, M. L.; SOTO, P. & CATENA, M. Characterization of the Campylobacter fetus subsp. venerealis adhesion to bovine sperm cells. Int. J. Morphol., 34(4):1419-1423, 2016. SUMMARY: Campylobacter fetus is extracellular bacteria of the genital tract of cattle. They cause infertility and abortion, but there is no documented information on the susceptibility of bovine sperm cells to this bacteria. The aim of this present work was to study the effects provoked by Campylobacter fetus subsp. venerealis when in interaction with bovine sperm cells. The bovine spermatozoa were obtained frozen bovine semen pooled from uninfected bulls, and were exposed to living campylobacter over different periods of time. Light microscopy and scanning electron microscopy first revealed a tropism, then a close proximity followed by tight adhesion between these two different cells. A decrease in the spermatozoa motility was observed. Motile bacteria were observed during the next 3 h, this process began with a tight membrane–membrane adhesion. The adhesion between Campylobacter fetus to the sperm cell occurred either by the flagella or by sperm head. Results from this study demonstrated with light microscopy scanning electron microscopy allowed us to characterize some aspects of the interaction of Campylobacter fetus subsp. venerealis and bovine sperm while preserving the cellular and bacterial structure. This ex vivo model might be useful for studies on adhesion and cytopathogenicity of different field strains of Campylobacter fetus. KEY WORDS: Adhesion; Campylobacter fetus subsp. venerealis; Sperm cells. INTRODUCTION Successful mammalian reproduction requires that pathogenesis of the bovine genital campylobacteriosis sperm migrate through a long and convoluted female (Chiapparrone et al., 2008; Chiapparrone et al., 2009; reproductive tract before reaching oocytes. For many years, Chiapparrone et al., 2011; Chiapparrone et al., 2014) intends fertility studies have focused on biochemical and to describe the behavior of a strain of Campylobacter fetus physiological requirements of sperm (Barth, 1995). There subsp. venerealis (C. fetus subsp. venerealis) bacteria of are a large number of microorganisms that propagate for the preputial smegma of bull in its interaction with sperm cattle seminal route. The pathogen can adhere to the sperm in the by light microscopy and scanning electron microscopy tail and/ or the head, or can infect the various components (Fletcher & Floodgate, 1973). of the seminal fluid as sperms, seminal plasma and leukocytes, among others) (Bielanski & Dubuc, 1993; Bielanski et al., 1994; Bielanski & Dubuc, 1994; Bielanski MATERIAL AND METHOD & Loewen, 1994; Bielanski et al., 2004). Many of the existing studies on the association pathogen-sperm are not conclusive because, the detection of this union is complex Bacterial strain. The C. fetus subsp. venerealis strain was and in some in some cases uncertain (Casadevall & Pirofski, isolated in beef bovine of Tandil - Argentina, from preputial 2000) as in the case of Campylobacter fetus (Bielanski et tract of Aberdeen Angus bull and identified using phenotypic al., 1994). test and PCR. The strain was grown for 48 h at 37 °C incubated under microaerophilic conditions which In the project of ex vivo model for the study of the correspond to the logarithmic growth phase. All experiments Laboratorio de Microbiología Clínica y Experimental, NACT. SAMP, Unidad Ejecutora de CONICET (CIVETAN). Facultad de Ciencias Veterinarias. Universidad Nacional del Centro de la Provincia de Buenos Aires, Argentina. 1419 CHIAPPARRONE, M. L.; SOTO, P. & CATENA, M. Characterization of the Campylobacter fetus subsp. venerealis adhesion to bovine sperm cells. Int. J. Morphol., 34(4):1419-1423, 2016. were performed with C. fetus subsp. venerealis-1 strain at a concentration of 1x109 cells/mL. Sperm cells. We used frozen bovine semen pooled from fertile bulls for artificial insemination. For interaction purposes, straws of frozen semen were thawed by immersion in a water bath at 37 °C for 30 s, centrifuged and the entire medium removed. The cells were washed once in PBS and transferred to Minimum Essential Medium Eagle (MEM) culture medium (M0643 Sigma-Aldrich) supplemented with 10 % fetal bovine serum. Viability tests were based on visual morphology and motility under dark field microscopy, each sperm cell was categorized as belonging to one of four motility categories (rapid progressive, slow progressive, non-progressive and Fig. 1. Campylobacter fetus subsp. venerealis adhered to head of immotile) and viability staining with Nigrosin-Eosin stain sperm cells for the apical end. Scanning electron microscopy. (Rodríguez-Martinez, 2013). 5500X. Adherence assay. For interaction analysis, suspensions of sperm cells were counted in a Neubauer chamber and exposed to C. fetus subsp. venerealis-1 in a cell ratio of 1:1 bacteria–sperm cells, for 3 h at 37 °C incubated under 5 % CO2. For control experiments, the same bacteria and sperm cells free were used. The bacterial motility can be determined under dark field microscopy. Giemsa stain. Sperm cells were fixed with acetone at 4° C and stained with Giemsa 10 %. Bacterial adhesion was observed and quantified by optical microscopy. Scanning electron microscopy. After checking the adhesion by Giemsa staining, innoculated cell cultures were processed Fig. 2. Campylobacter fetus subsp. venerealis adhered to head of according to Electron Microscopy Laboratory protocol of sperm cells for the apical end. Scanning electron microscopy. the College of Sciences, Universidad Nacional de Mar del 6000X. Plata (UNMdP). Briefly, the sperm cells were fixed with 3 % glutaraldehyde in sodium cacodylate buffer and washed with 0.1 M sodium cacodylate buffer. After dehydration with ethanol they were exposed to hexamethyldisilazane over- night, and then gold and palladium metallizing (Denton Vacuum Desk II). The samples were observed with scanning electron microscope (Jeol JSM-6460 LV). The system used was an EDAX Genesis XM4 - Sys 60, equipped with Multichannel Analyzer EDAX mod EDAM IV, Sapphire Si (Li) detector and super ultra-thin Window of Be, and EDAX Genesis version 5.11 software. RESULTS Fig. 3. Campylobacter fetus subsp. venerealis adhered to head and Of the sperm cells 78 % were alive, before bacteria - tail of sperm cells for the apical end. Scanning electron microscopy. sperm interaction. This number reduced to 56 % in the third 4000X. 1420 CHIAPPARRONE, M. L.; SOTO, P. & CATENA, M. Characterization of the Campylobacter fetus subsp. venerealis adhesion to bovine sperm cells. Int. J. Morphol., 34(4):1419-1423, 2016. hour, when bacteria and sperm cells were under interaction. the presence of one or more C. fetus subsp. venerealis adhered Sperm that were white (unstained) were classified as alive, by the apical body, mainly to the sperm head (Figs 1, 2 and 3), and those that showed any pink or red coloration were was observed and confirmed by routine colorations (Fig. 4). classified as dead, with the sole exception for sperm with a In others cases, it was possible to determine the presence of slight pink or red appearance restricted to the neck region bacterial shapes in an extracellular matrix, adhered to the (‘leaky necks’), which were assessed as live. Motile bacte- external membrane of the head and tail of sperm, grouping ria were observed during the next 3 h. with biofilm characteristics (Figs. 5 and 6). By scanning electron microscopy sperm cell treated with C. fetus subsp. venerealis showed adhesion and alterations in the bovine spermatozoa. All the superficial structures of spermatozoa such as head (66 %), mid piece, neck, and tail (34 %) were involved in bacterial adhesion. The capacity of adherence to different parts of the sperm, the number of adherent C. fetus subsp. venerealis and the characteristics of interaction were analyzed. In the images, Fig. 6. Observation of grouping of Campylobacter fetus subsp. venerealis adhered to head of sperm cells. Scanning electron microscopy. 7000X. DISCUSSION C. fetus subsp. venerealis is one bacteria that cause infertility in cows (Campero, 2002; Catena, 2002; Catena, 2003). However, there is no documented information about Campylobacter effect on sperm cells viability (Bhavsar & Fig. 4. Campylobacter fetus subsp. venerealis adhered to head of Kapadnis, 2006; Brenner et al., 2005; de Melo & Pechére, sperm cells. Giemsa stain. 100X. 1990; Graham & MacDonald, 1988; Joens et al., 2010) and there is no complete agreement on the detrimental role of bacteria presence in the semen. The ability of bacteria to bind to host cells is important as it represents an early event in the creation establishment in vivo microorganism - cell relationship (Casadevall & Pirofski, 2000; Benchimol et al., 2008; Chiapparrone et al., 2014). Sometimes, such binding process is also a requirement for pathogenicity. Understanding the capability of adherence of C. fetus subsp. venerealis to the host cell (Chiapparrone, 2011) is significant for a thorough knowledge of the initial steps involved in the pathogenesis of bovine genital campylobacteriosis. The light microscopy and scanning electron Fig. 5. Observation of more than one Campylobacter fetus subsp. microscopy allowed us to characterize